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1. INFECTION OF HOST CELLS BY DNA VIRUSES MICROBIOLOGY & IMMUNOLOGY ASSIGNMENT GROUP 4

2. VIRAL DNA REPLICATION Hepatitis B virus Parvovirus Adenovirus Herpesvirus Polyomavirus Papillomavirus Poxvirus

3. • Viruses must replicate their genomes to make new progeny • This always requires expression of at least one virus protein, sometimes many (hence always delayed after infection) • DNA is always synthesized 5’ – 3’ • Replication initiates at a defined origin ( Ori ) using a primer • The host provides other proteins

4. Requirements for DNA Replication • Ori recognition for initiation - binding to an AT-rich DNA segment • Priming of DNA synthesis - RNA - Okazaki fragments - DNA - hairpin structures - protein - covalently attached to 5’ end • Elongation • Termination

5. ` • Viruses don’t replicate well in quiescent cells • Induction of host replication enzymes and cell cycle regulators • Virus encoded immediate early and early gene products

6. • Small DNA viruses do not encode an entire replication system -encode proteins that orchestrate the host - Papillomaviridae , Polyomaviridae , Parvoviridae • Large DNA viruses encode most of their own replication systems - Herpesviridae , Adenoviridae , Poxviridae

7. Virus Encoded Proteins • Origin Binding Protein, Helicases and Primase • DNA polymerase and accessory proteins • Exonucleases • Thymidine kinase , RR, dUTPase

8. Replication Occurs at Replication Centers! • DNA templates and rep proteins • Form at discrete site ND10’s (PML bodies) • Polymerases, ligases , helicases , topoisomerases

9. Origin of initiation

10. Diverse viral genome structures

11. Origin Recognition Proteins • Polyoma Tag binds specifically to DNA • Papilloma E1 binds to ori in presence of E2 • AAV Rep68/78 binds at ends and unwinds DNA, also involved in terminal resolution • Adenovirus pTP binds at terminus and recruits DNA polymerase • Herpesvirus UL9 protein recruits viral proteins to AT-rich ori’s and then unwinds DNA

12. dsDNA Virus Genomes

13. ssDNA Genomes Circoviridae ITR ITR

14. Two Basic Modes of Replication Leading Leading Lagging

15. Leading vs. Lagging • Leading strand DNA synthesis is continuous • Lagging strand DNA synthesis is discontinuous • Direction of synthesis off of either template strand is the same

16. Protein Priming ITRs Single-stranded DNA Template Displaced ss Template DNA

17. The 5’-end problem

18. Semi-discontinuous DNA synthesis from a bidirectional origin No end problem!

19. Recognition and unwinding of SV40 origin Rp -A binds LT! T has 3’-5’ helicase ac.vity

20. Synthesis of leading and lagging strands Synthesis of long DNA Synthesis of RNA primers Synthesis of short DNA fragments Rf -C binds 3’OH along with PCNA and pol δ —RF-C a clamp loading protein —Allows entry of PCNA on DNA —Causes release of pol α Form sliding clamps along DNA

21. Synthesis continues…

22. Function of topoisomerases

23. SV40 large T • T is a species-specific DBP/OBP - Pre-initiation complexes do not form in the wrong species - Failure to interact with DNA pol α - primase • Binds and sequesters cell cycle regulators - Causes cells to enter S phase

24. Regulation of DNA synthesis • Most of our cells do not divide or do so rarely • Viruses do not replicate well in quiescent cells • Viruses must induce host replication proteins • Done by virus encoded immediate early and early gene products

25. Rb protein • Cellular retinoblastoma ( rb ) gene • Rb protein controls entry into S • Rb loss associated with tumors = tumor suppressor gene

26. Abrogation of Rb by viral proteins

27. DNA priming: Parvoviruses

28. Parvovirus DNA replication • Replication is continuous • No pol α, uses ITR to self-prime • Requires pol δ, RF-C and PCNA • Rep78/68 proteins are required for initiation and resolution: endonuclease , helicase , binds 5’-terminus • No replication fork, strand displacement No end problem!

29. Parvovirus DNA replication.. • When no helper adenovirus is present, Rep 78/68 level is low • Little viral DNA synthesis occurs • Genome integrates into host cell DNA • DNA replicates only in cells coinfected with helper adenovirus • Adenoviral helper proteins allow synthesis of large quantities of Rep 78/68